Effect of poly‐L‐lactic acid and polydioxanone biostimulators on type I and III collagen biosynthesis

Abstract Objective Safe, effective, and biocompatible minimally invasive procedures with the potential to stimulate collagen production have been made to recover dermal thickness and skin quality. The main of this animal model experiment was to observe the effect of poly‐L‐lactic acid (PLLA) and polydioxanone (PDO) biostimulators in collagen I and III after hypodermal injection. Methodology Sixteen adult female rats (Wistar) were randomized into four groups and had dorsal treatment with: G1: hypodermic subcision (HS) only; G2: HS and PLLA hypodermic injection (HI), G3: HS and PDO HI; G4: Control, with no treatment. Results In histochemical, it was observed hypodermal and dermal tissue in more organized thickness in G3 and in G4 when compared to G1 and G2. There was few difference in G1 compared to G4. The tissue of G2 showed irregularities in the arrangement of collagen fibers, less defined structure and lower distribution of type I collagen compared to the other groups. There is a greater tendency for the proportions of type III collagen among tissues treated with both biostimulators (G2 and G3). PLLA and PDO had relatively similar percentages of collagen when compared to G4. The amount of type I collagen was higher in tissues treated with subcision, while type III collagen was higher in tissues treated with both biostimulators. Conclusion G3 showed better performance in collagen production, although small, when compared with G2.

collagenases and, in this condition, there is no balance between collagen synthesis and degradation.Consequently, the disorganization of the remaining fibers is evident, apparently thinner, and more granular.These events lead to the replacement of type I collagen for type III collagen, found in younger and older individuals, respectively. 4,5broblasts play an important role in collagen synthesis and extracellular matrix organization participating in skin morphogenesis, angiogenesis, and healing.Through the discovery of the participation of gene expression modulators, such as transforming growth factor type β (TGF-β), it was possible to recognize its action in the expression of several genes of the extracellular matrix, serving as an adjunct in the coding of collagen I, III, IV, and V from the fibroblast. 6[14][15] Polydioxanone (PDO) is used as an absorbable material in the form of PDO threads to induce the formation of collagen. 16Recently, a new presentation of PDO powder is being marketed with the same indication of collagenesis activation; however, more detailed research on its performance in activating collagenesis and increasing dermal thickness and quality is still lacking.
To date, no experiments have been found comparing the effectiveness of PLLA and PDO biostimulators on skin collagenesis.In this way, the present study aimed to compare the effects of intradermal injection of PDO and PLLA in an animal model on the production of type I and III collagen in animal model.

METHODOLOGY
The experiment was approved by the Ethics Committee on the Use of Animals of the Ingá University Center (# 99/2020).

Experimental design
The flowchart summarizes the experimental steps of the study (Figure 1).

Biostimulators preparation
The reconstitution PLLA (Rennova Elleva, GANA R&D CO., Ltd, South Korea) and PDO (ULTRACOL, ULTRA V Co., Ltd, Korea) was performed with 14 mL of sterile distilled water under vigorous agitation for uniform powder mixing for 1 min every 10 min, until 1 h was completed.
After the agitation time, the products were submitted to rest for 4 h.
After the rest period, a new homogenization was performed, and the solution was used immediately.The remaining biostimulator solution was discarded.

Animals
Sixteen adult female rats (Wistar), weighing among 250-300 g, 4 The treatments were applied to the posterior dorsal region.The demarcation of the area to be treated (3 × 3 cm) was performed by tattooing Henna (Figure 2).Within this delimitation, a previous trichotomy was performed and then it was done general anesthesia of the animal (11.6% ketamine hydrochloride: Dopalen -50 mg/mL; and xylazine hydrochloride: Rompun -2 g/100 mL) at 0.3 mL/100 g of body mass in a ratio of 1:1 intraperitoneal PI.

Treatment
In the animals of G1, only subcision was performed in the hypodermic plane with a 22G cannula (Rennova); in groups G2 and G3, 0.5 mL of biostimulator (PLLA and PDO, respectively) was injected after hypodermic subcision.The animals of G4 did not undergo any intervention.
The animals were kept in laboratory conditions for 30 days until new anesthesia for tissue collection.

Euthanasia and material collection
Euthanasia was conducted under general anesthesia by administering ketamine hydrochloride 50 mg and xylazine 10 mg intraperitoneally, followed by exsanguination by cardiac puncture.Soon after, 3 × 3 cm of tissue, including hypodermic plane, was removed for histological evaluation (Figure 3).

Histological slides
Histological analysis was performed 30 days after subcision and injection of biostimulators.The tissue fragments were fixed in 10% buffered formaldehyde, dehydrated in solutions of increasing concentrations of alcohols (70%, 80%, 90%, and 100%), diaphanized in xylol and soaked in paraffin.The histological sections, with a thickness of 5 μm, were obtained in a microtome (American Optical, model 820) and stained by the hematoxylin-eosin (HE) technique, for evaluation of the general morphology, and Picrosirius Red for the detection of type I and III collagens. 17r staining in Picrosirius, the slides were dewaxed in 100% xylene, washed with 100% ethanol and in water.Then, the slides were submerged in 0.1% Sirius Red F3BA (BDH Laboratory Supplies, Poole, UK) in saturated picric acid for 1 h at room temperature.Washing was performed to remove excess dye and immersed in 0.1 N HCl for 2 min followed by another wash in water.Finally, the slides were dehydrated with ethanol and xylene and dried at room temperature until microscopic reading. 18

Microscopy and image capture
The histological slides were analyzed under an optical microscope (Nikon Eclipse, Shimjuku, Japan), coupled to a high-resolution camera (Nikon Ds-Fi1C, Shimjuku, Japan).
The digital images were captured in a specific program (NIS-Elements version 4.0, Prague, Czech Republic).To visualize collagen, the histological sections were visualized under polarized light, in the 40X objective, where the stained collagen fibers exhibited coloration from bright green to red. 18

Collagen quantification
The images obtained from the histological sections, stained with

Statistical analysis
Data normality was performed using the Kolmogorov-Smirnov test.
Intergroup comparison was performed with the analysis of variance (ANOVA) test to a selection criterion and Tukey's test.Statistical analysis was performed using the Statistic 12.0 software (Statsoft, Tulsa, Okla, USA), and the data were considered significant for p < 0.05.

RESULTS
The histological sections after 30 days of treatment with only subcision (G1), biostimulators (G2-G3), and the control group (G4) can be seen in Figure 4.
The HE stains showed that the tissue presents a more organized thickness in the treatment performed with the PDO biostimulator (G3) (Figure 4C) and in the control group (G4) (Figure 4D), when compared to the subcision (G1) (Figure 4A) and PLLA (G2) (Figure 4B) groups.
In the tissue of the experimental group with PDO (G3) (Figure 4C), it is possible to verify well-organized and compact collagen bundles.
Under polarized light, it was possible to verify with greater clarity the collagen fibers of the analyzed tissues.As observed, the tissue treated only with subcision using a 22G cannula (G1) (Figure 5A The distribution pattern of collagen fibers for this study showed a slight difference in collagen stimulation after subcision with a 22G cannula (G1) (Figures 5A and 6), since the tissue kept its tissue architecture practically intact compared to the control group (G4) (Figures 5D   and 6).On the other hand, the tissue treated with PLLA (G2) showed irregularities in the arrangement of collagen fibers, revealing a poorly defined structure and lower distribution of red color (type I collagen) compared to the other groups (Figure 5B).
All groups showed the presence of substantial green coloration (Figure 5A-D), which was confirmed after determining the proportions of green to red protein.However, the control group (G4) presented a lower percentage of type III collagen, green color (1062.90± 396) (Figure 6).
There is a greater tendency for the green proportions (type III collagen) among tissues treated with PLLA (G2) and PDO (G3) biostimulators (Figure 6), and there seems to be a relationship between biostimulator and higher biosynthesis of type III collagen in the recent collagen synthesis in this study.
In the 30-day period, type I collagen was higher with subcision and in the control group versus PLLA (G2) and PDO (G3) (p = 0.000).In the same period, the staining for type III collagen was higher with PDO, while the PLLA showed a similar type III collagen area (p = 0.000).
Collagen values differ from each other when statistically compared between the different types of treatment and control after 30 days of intervention (Table 1).

DISCUSSION
To the best of our knowledge, this study is an unprecedented research.
It was demonstrated that the percentage of type I collagen is similar between the control group and the tissues treated with subcision using a 22G cannula, reflecting a significant difference in the percentage of type III collagen, 1060.90 and 1590.24(p = 0.000), respectively.
Despite these findings, the quantification of collagen for subcision did not promote an expressive formation of collagen fibers when compared with the control group.Therefore, in the eyes of the evidence, little difference in skin quality will be achieved with subcision.
Therefore, caution should be exercised when opting for the subcision technique with a single treatment modality, as suggested by other studies using this technique for the treatment of wrinkles and depressed scars. 19e small difference in collagen percentages between the experimental groups may be related to the analysis time, since the collagen stimulus is time dependent.That said, the results of the collagen stimulus may not be visualized for weeks, and it is necessary to wait for the results individually, because the response and the degree of improvement of the skin after the biostimulus will depend intrinsically on the characteristics of each patient, age, sex, and quality of the skin.From this perspective, it is important that professionals qualified in injectable procedures on facial esthetic have this knowledge so as not to reproduce overcorrections with additional treatments early.In addition, when performing the treatment with collagen biostimulators, the patient should be advised that the result will be achieved more than 30 days after the injection of the biostimulator. 21 tissues mature, new collagen fibers are synthesized, increasing the percentage of total collagen.In addition, the green-colored collagen fibers become thicker, replacing them with red-colored fibers, thus increasing the areas of occupation of type I collagen. 18is mechanism may justify the trends in green-to-red protein ratios in our experimental trials (Figure 6).Possibly, the largest proportion of green-stained proteins of groups G2 (PLLA) and G3 (PDO) are newly synthesized collagen fibers induced by biostimulators.Since the effect of biostimulators are late and gradual, being perceived by the dermal thickening promoted through collagenesis, forming type I collagen and in a smaller amount, type III collagen. 7e inflammatory reaction after the injection of collagen biostimulators initiates the deposition of type III collagen fibers around the microspheres of the material, followed by a fibroblastic tissue response and peripheral type I collagen deposition.This mechanism continues for weeks until there is remodeling of type III collagen with predominance of type I collagen in the newly formed tissue, resulting in improved dermal thickness and quality. 22,23udies on collagen synthesis from the injection of PLLA into the dermis of Wistar rats 24 revealed that the biostimulator was able to stimulate the production of high content of type I collagen compared to the control group.This efficient behavior justifies its use to attenuate imperfections by stimulating collagen production in the skin. 24wever, in this study, the area occupied by type I collagen for the same biostimulator was smaller than that of the control group.Once again, we reinforce the hypothesis that perhaps the experiment time was insufficient to determine the total potential of the biostimulator.Therefore, it is suggested that further in vivo studies with animal models be developed to test this hypothesis.
In Brazil, PDO is marketed in the form of smooth threads to stimulate collagen production and as spiculated wires promoting liting and facial rejuvenation.Smooth PDO wires act as a foreign body, stimulating the formation of subcutaneous fibrous tissue, which results in improved skin tension. 25Unlike smooth wires, the effect of spiculated wires is immediate, since it acts as a vertical vector for repositioning the soft tissues of the face. 26On the other hand, little is known about biocompatibility, safety, and efficacy of PDO powder.For this reason, this material was investigated in this study.
An investigation on the biostimulatory effect of the recently developed PDO 27 revealed that PDO microspheres demonstrate a similar effect regarding collagenesis and inflammatory response when compared with PLLA and polycaprolactone (PLC).In addition, PDO also The technique of subcision and injection of biostimulators in the intradermal plane may have been one of the reasons for the equivalent amounts of collagen between the experimental groups.Histological studies also developed in animal models comparing collagen production after intradermal and subdermal injection of biostimulators revealed more significant amounts of collagen by the intradermal injection technique. 12Thus, these results serve as a basis for professionals to apply biostimulator intradermally to achieve better results, durability, and patient satisfaction.
Finally, the limitations of this study can be presented by the small number of animals used in the experiments and by the type of study model (in animals).On the other hand, the data show that the animal tissues treated with biostimulators revealed a greater presence of type III collagen, which was later replaced by type I collagen. 28 this sense, further randomized clinical studies are needed to determine whether this process of collagenesis occurs in humans after the injection of the biostimulators investigated, and whether these bring real benefits to esthetic treatments of collagen stimulation, improving dermal quality, in addition to determining the level of safety and efficacy of the biostimulators PLLA and PDO powder.

CONCLUSION
The performance of the biostimulators revealed that the PDO resulted in a more active collagenesis, stimulating greater production of collagen types I and III.The results of this study suggest that in the first month after treatment complete remodeling may not occur for the formation of new collagen fibers, which may have influenced the results regarding the type and amount of newly formed collagen when comparing the four experimental groups.Therefore, further studies are needed to determine the total performance of the biostimulators investigated in a longer period.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in CV LATTES at http://lattes.cnpq.br/2135059544500650.
Picrosirius-Red, were analyzed with the software Image-Pro Plus (version 4.5 -Media Cybernetics).We evaluated 10 images per animal/group.In each image, the area occupied by type I collagen (red) and type III collagen (green) was determined separately.The results were F I G U R E 2 3 × 3 cm area demarcation in the posterior dorsal region for the treatment.F I G U R E 3 Tissue excision 30 days after experiment.expressedas the area occupied by each type of collagen (in percentage) in relation to the total area analyzed.18 ) presented thick fibers that were predominantly yellow and red, indicating the presence of type I collagen.The green fibers were evidenced in greater intensity in the tissues treated with biostimulators (Figure 5B, C: PLLA -G2; and PDO -G3, respectively), indicating the presence of type III collagen.It was also possible to notice that greater distribution of green fibers (indicative of type III collagen) was located in the tissues treated with PDO (G3) (Figure 5C).In addition, in Figure 5C it is possible to visualize the PDO microparticles in blue spherical format, indicating the induction of collagen formation in the region.

F I G U R E 4 TA B L E 1
Tissue sections of the posterior dorsal region after 30 days of subcision, application of biostimulators and control group in HE stains.(A) Subcision with 22G Cannula (G1); (B) Subcision and treatment with 0.5 mL of PLLA in a single session (G2); (C) Subcision and treatment with 0.5 mL of PDO in a single session (G3); (D) Control with no treatment (G4).HE, hematoxylin-eosin; PDO, polydioxanone; PLLA, poly-L-lactic acid.Intergroup comparison for collagen production (ANOVA to a selection criterion and Tukey's test).

F I G U R E 5
Tissue sections of the posterior dorsal region of mice after 30 days of subcision, application of biostimulators and control group in Picrosirius staining and visualized under polarized light.(A) Subcision with 22G Cannula (G1); (B) treatment with 0.5 mL of PLLA in a single session (G2); (C) treatment with 0.5 mL of PDO in a single session (G3); (D) control (G4).PDO, polydioxanone; PLLA, poly-L-lactic acid.

F I G U R E 6
Proportion of red (type I collagen) green (type III collagen) staining in tissue sections of the posterior dorsal region of mice after 30 days of subcision, application of biostimulators and control group.G1: Subcision with 22G Cannula; G2: treatment with 0.5 mL of PLLA in a single session; G3: treatment with 0.5 mL of PDO in a single session; G4: control.The results are presented as average and standard deviation.PDO, polydioxanone; PLLA, poly-L-lactic acid.showedbetter degradability and significant decrease in skin roughness for the study.In this sense, these results, although incipient, open new avenues for the development of randomized clinical studies in humans to confirm in the future the safety and efficacy of the recently developed PDO powder-based biostimulator, aiming at its use in clinical practice as a biostimulator of elastic and collagen fibers.
Different letters on the same line indicate the presence of a statistically significant difference between the group indicated by the Tukey's test.ANOVA, analysis of variance; SD, standard deviation.*Statistically significant for p < 0.05.